Oreochromis spp.) via oral administration as a feed additive. We demonstrated that nanotechnology-based drug delivery systems represent a novel and promising strategy for improving the oral absorption of these androgen hormones, resulting in a 50% w/w reduction in the hormonal dosage during the sex reversal period. Therefore, we strongly believe that this newly optimized nanotechnology is one of the solutions for sustainable aquaculture production.Lipid-based nanocarriers have proved effective for drug and hormone delivery. The androgen hormone 17 alpha-methyltestosterone (MT) is used for sex reversal in tilapia; however, the hormone has biopharmaceutical limitations such as insolubility, low stability, limited intestinal absorption, and poor bioavailability. Incorporating MT into lipid-based nanocarriers to improve hormone uptake in tilapia is an interesting prospect. In the present study, we reported an improved version of alkyl polyglucosides loaded with 17 alpha-methyltestosterone integrated into nanostructured lipid carriers (APG-NLC) for the masculinization of red tilapia (b < 3), with a relative condition factor (Kn) of more than 1. Therefore, MT-APG-NLC (30 ppm) would seem to be a promising, cost-effective way to reduce the dose of MT used for the masculinization of farmed red tilapia.The aim of the present study was to optimize a masculinization platform for the production of all-male red tilapia fry by oral administration of 30 and 60 ppm of MT and alkyl polyglucoside nanostructured lipid carriers (APG-NLC) loaded with MT, respectively, for 14 and 21 days. The characterization, encapsulation efficiency and release kinetics of MT in lipid-based nanoparticles were assessed in vitro. The results showed that the MT-loaded nanoparticles were spherical, ranging from 80 to 125 nm in size, and had a negative charge with a narrow particle distribution. The APG-NLC loaded with MT provided higher physical stability and encapsulation efficacy than the NLC. The release rate constants of MT from MT-NLC and MT-APG-NLC were higher than those of free MT, which is insoluble in aqueous media. There was no significant difference in survival between the fish administered MT or the those fed orally with MT-APG-NLC fish. According to the logistic regression analysis, the sex reversal efficacy of MT-APG-NLC (30 ppm) and MT (60 ppm), resulted in significantly higher numbers of males after 21 days of treatment compared with the controls. The production cost of MT-APG-NLC (30 ppm) after 21 days of treatment was reduced by 32.9% compared with the conventional MT treatment group (60 ppm). In all the treatments, the length–weight relationship (LWR) showed negatively allomeric growth behavior ( Oreochromis spp.) is recognized as an important farmed fish species worldwide because it can easily adapt to the environment and has a fast growth rate [Red tilapia  have been developed and used for drug and hormone delivery ,12. The Oreochromis spp.) cultures. Therefore, the aim of the present study was to optimize a ready-to-use masculinization nanoparticle that could be delivered orally to red tilapia fry to reduce the dose and duration of MT administered orally to fish. We characterized four classes of lipid-based nanoparticles, including solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) with and without the integration of alkyl polyglucoside (APG), from which we selected a promising nanocarrier platform to use as an alternative for the oral delivery of MT in a sex reversal study in red tilapia fry.The potential of lipid-based nanocarriers to enhance the sex reversal efficacy of MT in fish has not been investigated. Designing a lipid-based nanodelivery system containing 17 alpha-methyltestosterone (MT) could offer a promising masculinization platform to induce single-sex red tilapia . Medium-chain triglycerides (MCT) and ethoxydiglycol were purchased from PC Intertrade . Span 80, Tween 20, glycerol and Synperonic PE/F68 were purchased from Croda . Montanov 82 was purchased from Chemico . All chemicals, materials and water were purified and of laboratory grade. Alkyl-poly glucosides (APG) were purchased from Croda .The NLC and SLN, the subtypes of the lipid-based nanoparticles, were fabricated using hot high-energy homogenization techniques . As showThe oil mixture was dissolved and mixed with the oil phase surfactant by stirring at 200 rpm and 70 °C on a hotplate. Ethoxydiglycol (3 g), cetearyl alcohol and cocoglucoside (1 g), and Span 80 (3 g) were added to the oil mixture and stirred until completely dissolved. An aqueous phase mixture was prepared by mixing purified water, Tween 20 (3 g), glycerol (2.5 g) and Synperonic PE/F68 (2 g) on a hotplate stirrer, and the mixture was poured into the lipid phase. A high-speed homogenizer  at 10,000 rpm was used to prepare the pre-emulsion for 5 min. Furthermore, the mixtures were sonicated in a sonicator unit  at a 20 amp pulse of 30 s on and off at 5 s intervals for 5 min to prepare the testosterone–nanostructure lipid nanocarriers (MT-NLC). Testosterone–solid lipid carriers (MT-SLN) were also fabricated by following the same preparation steps as for NLC by without adding MCT (the liquid lipid). The MT-NLC and MT-SLN were cooled to an ambient temperature and used for subsequent studies. For the APG-integrated lipid-based nanoparticles, APG was added to a lipid mixture to fabricate alkyl-polyglucoside-integrated nanostructured lipid carriers (MT-APG-NLC) and alkyl-polyglucoside-integrated solid lipid nanoparticles (MT-APG-SLN).The hydrodynamic diameter, particle distribution and particle surface charge of lipid-based nanoparticles were characterized by dynamic light scattering (DLS) using a zetasizer . DLS experiments were performed using a He-Ne laser . Before the measurements, the samples were diluted 50 times in purified water. All reported data were from experiments performed in triplicate at 25 °C. The particles’ stability was detected using changes in the particles’ size and the particles’ phase separation after incubation under various storage conditions  for two months.w/w), dried in a dry cabinet overnight and observed under 100 kV with a magnification of ×25k and ×100k. The particles’ size and morphology were determined for an average of more than 50 particles for each sample.The particles’ size and morphology were characterized using a transmission electron microscope . Samples were diluted 50-fold in deionized water, dropped onto a carbon grid, stained with phosphotungsten (1% v/v) and a flow rate of 1.0 mL/min. The encapsulation efficiency (EE) of testosterone in lipid-based nanoparticles was calculated by using the following equation [iC corresponds to the initial concentration of testosterone added to LBN particles and fC represents the concentration of free testosterone.The encapsulation efficiency (EE) was evaluated using an Amicon centrifuge filter . The lipid-based nanoparticles (10 mL) were loaded into the filter membrane and centrifuged  by following the manufacturer’s protocol. The supernatant was filtrated through a syringe filter , referred to as the “non-encapsulated drug concentration”. Furthermore, the encapsulated lipid particles were extracted using the liquid extraction method and filtrated through a syringe filter  to be used as the encapsulated drug concentration . High-peequation .EE (%) R corresponds to the retention of MT at time (t), and n and k correspond to the release mechanism and release rate constants, respectively.The release profiles of MT from NLC and APG-NLC were determined and compared with those of free testosterone (diluted in ethanol) using a dialysis membrane . The samples were placed in a dialysis membrane and incubated in the released buffer . The released buffer was collected and replaced with fresh released buffer at regular intervals. The concentrations of testosterone were determined using HPLC-PAD as described above. Moreover, Avrami’s equation was used to determine the release mechanism and release constant of MT-synthesized-LBN.The protocols for the fish experiments used in this research were approved by the animal ethics committee of Chulalongkorn University Animal Care and Use Committee .1 (30 ppm of MT in the feed for 14 days), T2 (30 ppm of MT in the feed for 21 days), T3 (60 ppm of MT in the feed for 14 days), T4 (60 ppm of MT in the feed for 21 days), Control 2 , T5 (30 ppm of APG-NLC loaded with MT in the feed for 14 days), T6 (30 ppm of APG-NLC loaded with MT in the feed for 21 days), T7 (60 ppm of APG-NLC loaded with MT in the feed for 14 days) and T8 (60 ppm of APG-NLC loaded with MT in the feed for 21 days) with four replications. The red tilapia fry were distributed into 100 L fiber tanks containing dechlorinated fresh water under continuous aeration. The water (50%) was exchanged daily, and the tanks were replenished with fresh dechlorinated water. The experiment was performed at a controlled temperature within the range of 25–33 °C for air temperature and 25–28 °C for water temperature. The pH and content of dissolved oxygen (DO) were also determined to be within the range of 7.4–8.2 and 5.0–6.0 mg/L, respectively.The red tilapia fry (8-day-old swim-up (Stage 5)) were obtained from a hatchery unit from a GMP Certified fish farm in Ayutthaya Province, Thailand. Furthermore, the red tilapia swim-up fry were acclimatized (3 days) and randomly divided into 10 treatments (400 fish/treatment). The experimental groups were Control 1  9931, mixed with ethanol (ET) without MT), T1, T3, T5 and T7) and 21 days . At the end of 21 days of feeding, the fish in each group were counted, and the body weight was recorded to indicate the growth performance of the red tilapia fry.To prepare the MT-ET diet, MT was diluted in ethanol, sprayed, mixed with the commercial diet and air-dried. In addition, the MT-APG-NLC diet was prepared by directly mixing the APG-NLC loaded with MT and a commercial diet. Fish in all groups were fed at 10% of body weight five times per day. The fish were fed with a commercial diet until the 45th day of the trial, after completing the sex reversal treatment for 14 days (Ta is the rate of the change in weight with a constant length and b is the weight of one unit of length (slope) estimated from the linear regression equation transformed by taking the natural logarithm (log) of both sides.b = 3, an isometric pattern of growth occurs but when b is >3 or <3, an allometric pattern emerges, which may be positive when the length increases relative to the body’s thickness or negative when the length increases relative to the body’s thinness [The length–weight relationship (LWR) between the total length and body weight was expressed as W = aLb(thinness .n) was developed for assessing the health condition of tilapia under all the treatments.o is the observed weight and Wc is the calculated weight [n > 1, while the organism was in poor growth condition compared with an average individual of the same length when Kn < 1.The relative condition factor . The effects of the treatments on different parameters were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc tests for multiple comparisons. The effect of dietary supplementation with MT and MT-APG-NLC on the sex ratio  of red tilapia fry was analyzed using a binary logistic regression model. Linear regression analysis was used to assess the length–weight relationship. A value of The size of all four synthesized nanoparticles was lower than 125 nm. APG-SLN (122.23 nm) and APG-NLC (84.21 nm) were both smaller than bare SLN (125.30 nm) and NLC (94.56 nm). The average surface charge and polydispersity index (PDI) of the synthesized nanoparticles were lower than −20 mV and 0.3, respectively, as shown in However, under accelerated (45 °C) conditions, the size of the particles tended to increase to 144.83 nm without phase separation and sedimentation. For the bare NLC particles, the size was increased, and phase separation was observed under accelerated (45 °C) conditions. In addition, TEM images showed the spherical particles of LBNs within the range of 90–120 nm A–D.Chemical characterization using HPLC demonstrated that the encapsulation efficiency (EE) values of the synthesized lipid-based nanoparticles were 70.52 ± 3.42%, 70.06 ± 2.24%, 83.91 ± 4.01% and 84.75 ± 13.56% for MT-SLN, MT-APG-SLN, MT-NLC and MT-APG-NLC, respectively. On the basis of the stability and encapsulation efficiency of the synthesized particles, MT-NLC, MT-APG-NLC and MT-ET were selected for evaluation in an in vitro release study. During the first three hours, MT released from MT-NLC, MT-APG-NLC and free MT (dissolved in EtOH) showed release percentages of 16.9 ± 1.0%, 14.1 ± 2.0% and 11.6 ± 2.4%, respectively. A gradual release of MT from MT-NLC, MT-APG-NLC and MT-ET was observed, which reached 38.6 ± 2.5, 30.9 ± 4.1 and 11.4 ± 1.8% within 8 h and extended to the maximum in 24 h A.n) and release rate constant (k)  B.n) were found to be close to 1, being 0.95 for MT-NLC and 1.12 for MT-APG-NLC. Simultaneously, the release mechanism (n) of MT-ET was classified in two separate steps: 0.36 for the first two hours and 0.04 after three hours. Moreover, the release rate constant of MT from MT-NLC and MT-APG-NLC was found to be 0.587 × 10−1/h and 0.637 × 10−1/h, respectively. The release constant of MT-ET was lower than 0.01 × 10−1/h, as shown in The release mechanisms  between the effects of the concentrations of MT and MT-APG-NLC and the duration of the sex reversal period on the male ratio of red tilapia. The logistic regression model found that the male percentage in T1 , T2 , T4 , T5 , T6  and T8  was significantly higher than that in Control 1, Control 2, T3 and T7 , which was significantly higher (p < 0.05) than that of all the other experimental groups. The chi-square analysis of Control 1  and Control 2  indicated that males and females were equally represented. Likewise, there was no significant change in the normal 1:1 sex ratio in fish fed with T3  and T7 . All of the studied fish were found to be either male or female.The Tp > 0.05) compared with the control groups . Furthermore, the final body weight of red tilapia was also measured in the group that received the sex reversal diets for 21 days. The final body weights of red tilapia were similar among the controls, MT-APG-NLC (30 ppm), MT-APG-NLC (60 ppm), MT (30 ppm) and MT (60 ppm) groups  groups . There wb-values were lower than 3.0 for all treatment groups, which indicated negatively allomeric growth behavior and implied that the length of tilapia increased relative to body thickness. The higher r2 values (>0.95) obtained in the assessment of LWR under all the treatments suggested a good quality of fit for the predictions of the linear regression. Furthermore, a significant correlation (p < 0.05) was observed between the length and weight under all the treatments. The relative condition factor (Kn) was higher than 1.0, demonstrating that the fish were in good condition during the treatment period.The length–weight relationship was used to determine the growth performance of hormone-treated tilapia . The calMethyl testosterone is unlikely to have a detrimental effect on the flesh of fish after the sex reversal treatment (SRT) has been discontinued; nonetheless, its effects on the major organs of fish, the metabolic profiles and nucleic acids are inconclusive . HoweverCyprinus carpio [Lipid-based nanocarriers have been shown to be a landmark in drug and hormone delivery. The application of LBN loaded with steroid derivatives, such as exemestane, spironolactone or methylprednisolone aceponate, has been developed and approved to enhance their in vivo bioavailability ,36,37. Ns carpio . In our s carpio . APG hass carpio ,41. In ts carpio . The sols carpio . NLC pars carpio ,45,46. Is carpio ,48. The s carpio .Furthermore, an in vitro investigation showed that the amount of MT released, which was dissolved in ethanol from 3 h to 24 h, could be classified into two separate steps. In both steps, the values of the release mechanism were lower than 1, which suggested that the MT was released through a diffusion mechanism, while the MT loaded in lipid-based nanoparticles was released by first-order release kinetics . The ratdmrt1 [4 treatment produced 89.7% sex-reversed males in red tilapia. Our finding is in contrast with previous studies that reported 100% males in commercial tilapia farms [6 group produced 98.5% males, which is a significantly higher percentage of male tilapia compared with previous studies that found less than 70% sex-reversed males after the administration of 30 mg/kg of conventional MT for 28 days [dmrt1, an important mechanism underpinning gonadal masculinization in red tilapia that requires further investigation. The higher dose of MT-APG-NLC (60 ppm) resulted in a significant decrease in the percentage of males and their growth, which may be due to a reduction in the intake of feed. Moreover, the reduction in the percentage of male tilapia as the concentration of MT-APG-NLC increased could possibly be due to paradoxical feminization and the aromatization of MT to estradiol, which expresses the female sex hormone [The conventional sex reversal treatment (MT) has the potential to disrupt the natural differentiation process, even after its initiation, by interfering with normal embryonic gene expression patterns and physiological regulation, resulting in a skewed sex ratio towards males . Moreovedmrt1 ,53,54. Iia farms ,56, whic 28 days . Our fin hormone ,59. Thes6 had the lowest production costs, indicating that the MT-APG-NLC treatment is a more cost-effective alternative to the conventional sex reversal treatment for producing all-male red tilapia fry.The conventional sex reversal treatment of tilapia results in a varied survival rate of fish ,61,62,63The negative allometric growth showed that the LWRs of the control group were in good agreement with those of the MT treatment group. Similarly, Nile tilapia and red tilapia showed negative allometric growth behavior ,65. ImpoAPG-integrated lipid-based nanoparticles are a new LBN platform for delivering the androgen hormone MT as a sex reversal agent to tilapia via dietary administration. APG-NLC achieved high encapsulation efficiency, thermal stability and cost-effectiveness for the masculinization of tilapia with MT when delivered orally at 30 ppm for 21 days. This platform is a promising way to reduce the dose of artificial sex reversal hormones used in red tilapia aquaculture for masculinization.


Lamotrigine is an effective antiseizure medication that can be used in the management of focal and generalized epilepsies in pediatric patients. This study was conducted to quantify and compare the solubility of lamotrigine in age-specific biorelevant media that simulated the fasted and fed conditions of the gastric and intestinal environments in pediatrics and adults. Another aim was to predict how traditional, re-formulated, modified, and new oral formulations would behave in the gastric and intestinal environments across different age groups.Solubility studies of lamotrigine were conducted in 16 different age-specific biorelevant media over the pH range and temperature specified by the current biopharmaceutical classification system-based criteria. The age-specific biorelevant media simulated the environments in the stomach and proximal gastrointestinal tract in both fasted and fed conditions of adults and pediatric sub-populations. The solubility of lamotrigine was determined using a pre-validated HPLC-UV method.Lamotrigine showed low solubility in the 16 age-specific biorelevant media as indicated by a dose number of > 1. There were significant age-specific variabilities in the solubility of lamotrigine in the different age-specific biorelevant media. Pediatric/adult solubility ratios of lamotrigine fell outside the 80-125% range in 6 (50.0%) and were borderline in 3 (25.0%) out of the 12 compared media. These ratios indicated that the solubility of lamotrigine showed considerable differences in 9 out of the 12 (75.0%) of the compared media.Future studies are still needed to generate more pediatric biopharmaceutical data to help understand the performances of oral dosage forms in pediatric sub-populations.The online version contains supplementary material available at 10.1186/s12896-023-00809-2. The biopharmaceutical classification system (BCS) has been used to support and hasten the development of traditional, new, and/or modified immediate-release solid oral dosage forms (IRSODFs) . Drug reThe permeability and solubility of APIs can vary by age, developmental status, and maturation. Recently, there has been a growing interest in studying how the permeability and solubility of APIs can change using age-specific criteria for the volume and constituents of the gastrointestinal fluids , 13–18. Since its approval in different countries, lamotrigine has become one of the most commonly used antiseizure medications. Lamotrigine is one of the first-line treatments for Lennox-Gestault syndrome, focal-onset tonic-clonic seizures, and simple and complex partial seizures , 26. In Traditional, re-formulated, and novel IRSODFs containing lamotrigine as the API are needed for adult and pediatric patients. A recent study explored the possibility of extrapolating adult lamotrigine solubility from adults to children . HoweverAcetonitrile, egg lecithin, sodium hydroxide, acetic acid, pepsin, maleic acid, glyceryl monooleate, hydrochloric acid, sodium oleate, sodium taurocholate, sodium hydroxide, sodium chloride, sodium acetate, and methanol were obtained from Sigma-Aldrich Chemie GmbH . Lamotrigine was a gift from Jerusalem Pharmaceuticals Co. Ltd. HPLC grade water was used in this study. Dialysis tubes with molecular weight cutoffs of 12,000–14,000 Da were bought from Medicell Membranes Ltd., London, UK. For the preparation of all biorelevant media, ultra-pure (Milli-Q) water was used. Adult fed-condition biorelevant media were prepared using whole cow milk. The cow milk was pre-treated using ultra-high temperature and contained < 4% fat. The pediatric fed-condition media were prepared using three infant formulas: (1) cow milk-based formula , (2) soya-based formula , and (3) phenylalanine-free formula for infants and toddlers with phenylketonuria . The instruments used included Agilent Technologies 1200 series HPLC system , pH meter, and borosilicate glass tubes.The 16 different age-specific biorelevant media that simulated the environments in the stomach and proximal gastrointestinal tract in fasted and fed conditions of adults and pediatric sub-populations were prepared as previously described , 12, 32.Solubility studies were performed to assess the solubility of lamotrigine in the 16 different age-specific biorelevant media.To each of the 16 age-specific biorelevant media, excessive amounts of lamotrigine that were sufficient to oversaturate 10 mL were added in borosilicate glass tubes , 12, 24.The fed-conditions media contained milk or formula. Because these media existed as complex multiphase systems, the presence of proteins would impede the filtration of these samples through the 0.45 μm filters , 33. The0) which was informed by the volume of the glass of water with which IRSODFs are administered has been considered as one of the most important variables that affects the behaviors of IRSODFs [0 of 250 mL was used to compute the dose number (D0) of lamotrigine in adults.In IVBESs, IRSODFs are swallowed by adult volunteers with the help of a glass of 250 mL of water , 34. The IRSODFs , 11, 12.0 values were used to calculate the age-specific D0 values for (1) neonates (birth to 27 days) [Age-specific V27 days) , (2) inf27 days) , 11. It 27 days) , 38. Con27 days) –41. When27 days) , 9, 11. 27 days) .0 values were calculated as previously described [0 values are shown in Supplementary Table To obtain the median weights of neonates, infants, and children, the growth charts developed by the Centers for Disease Control and Prevention (CDC) were used . The medescribed . The med0 values in pediatric and adult populations were used to assign lamotrigine to either a high or low solubility class. If Lamotrigine’s highest dose strength is soluble in a volume equivalent to or less than the age-specific V0 value across various pediatric sub-populations, and over a pH range of 1.2–6.8 at 37 ± 1 °C, then it would be categorized as having a high solubility class. Otherwise, lamotrigine was said to have a low solubility class.Solubility values of lamotrigine in the age-specific VLamotrigine was determined using an HPLC-UV method as previously described in the US Pharmacopeia . The mob0 values were calculated as previously described using the age-specific V0, maximal dose strength, and saturated solubility [Maximal dose strengths of lamotrigine were obtained from the British National Formulary (BNF) 78 (Sep 2019 to Mar 2020) . Doses flubility , 46.n-octanol/water partition coefficient (LogP) were obtained from the DrugBank database [n-octanol/water partition coefficient (cLogP). A correlation plot was created between LogP and cLogP for a set of 35 drug molecules including metoprolol that was used as a benchmark for high/low benchmark of paracellular intestinal permeability from [The molecular descriptors and other physicochemical properties of lamotrigine like the experimentally determined water solubility and the database . Additioity from , 48–50. pediatric/µadult × 100%). When the ratio was < 100%, this indicated inferior solubility of lamotrigine in the pediatric media compared to that in the adult media. On the other hand, when the ratio was > 100%, this indicated superior solubility of lamotrigine in the pediatric media compared to that in the adult media [The data obtained in this study were entered and handled into GraphPad Prism for Windows v.7.0. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests was used to detect differences in the solubility of lamotrigine in the different age-specific biorelevant media. Pediatric/adult solubility ratios were computed as percentages by dividing the solubility of lamotrigine in the age-specific pediatric media by those in the equivalent adult media  compared to that in fasted-state simulated intestinal fluid and pediatric fasted-state intestinal media formulated with bile salt concentrations 50% of the adult levels. Irrespective of the type of feed, the solubility of lamotrigine in fed-conditions simulated gastric fluid was found to be significantly higher  than that in pediatric fed-conditions gastric media. Furthermore, in infant (1–12 months) fed-conditions, the solubility of lamotrigine was significantly higher  in pediatric fed-conditions intestinal media representative of milk-based formula and fed-conditions simulated intestinal fluid than that in neonate fed-conditions intestinal media representative of breast milk and milk-based formula.D0 values in the corresponding biorelevant media are ≤ 1. Conversely, APIs are assigned a low solubility class when their D0 values in the corresponding biorelevant media are > 1. The calculated D0 values of lamotrigine in all age-specific pediatric and adult biorelevant media. These values indicated that lamotrigine had low solubility. The calculated D0 values of lamotrigine in all age-specific biorelevant media are shown in Table According to the BCS criteria, APIs are assigned a high solubility class when their The pediatric/adult solubility ratios of lamotrigine are shown in Fig. 2. Metoprolol was previously used as a benchmark for low/high intestinal permeability. The experimentally determined LogP was 2.15 and the polar surface area of metoprolol was 50.72 Å2. ALOGPS predicted a cLogP of 1.80 and ChemAxon predicted a cLogP of 1.76 for metoprolol. Lamotrigine cannot be conclusively assigned to either a high or a low permeability class. Previous studies classified APIs to a low permeability class when they had LogP values higher than that of metoprolol. Similarly, APIs were assigned to a low permeability class when they had a polar surface area of less than 90.0 Å2.The experimentally determined LogP of lamotrigine was 1.93. ALOGPS predicted a cLoP of 1.87 and ChemAxon predicted a cLogP of 1.93 for lamotrigine. The polar surface area of lamotrigine as predicted by ChemAxon was 90.71 ÅPrevious studies reported conflicting bioavailability values for lamotrigine in humans. While some studies reported bioavailability values approaching 100% , 53, oth2. This indicated that lamotrigine had low permeability. Experimentally determined LogP values of 36 drugs were plotted against their corresponding cLogP showed excellent correlation  –23, 48. − 6 cm/s . Moreove− 6 cm/s . HoweverThe main limitation of this study is that the age-specific media were adopted and modified from the literature , 12. It The findings of this study demonstrated that lamotrigine has low solubility in the age-specific biorelevant media for adults and different pediatric sub-populations. The study also showed significant differences in the solubility of lamotrigine in different age-specific biorelevant media and IRSODFs containing lamotrigine could exhibit significant variability in their in vivo performance. Therefore, the current BCS-based criteria might not be directly applied in pediatric sub-populations. Future studies are still needed to generate more pediatric biopharmaceutical data to help understand the performance of IRSODFs in pediatric sub-populations.Below is the link to the electronic supplementary material.Supplementary Material 1


Quantitative image analysis of cancers requires accurate tumor segmentation that is often performed manually. In this study, we developed a deep learning model with a self-configurable nnU-Net for fully automated tumor segmentation on serially acquired dynamic contrast-enhanced MRI images of triple-negative breast cancer. In an independent testing dataset, our nnU-Net-based deep learning model performed automated tumor segmentation with a Dice similarity coefficient of 93% and a sensitivity of 96%.Accurate tumor segmentation is required for quantitative image analyses, which are increasingly used for evaluation of tumors. We developed a fully automated and high-performance segmentation model of triple-negative breast cancer using a self-configurable deep learning framework and a large set of dynamic contrast-enhanced MRI images acquired serially over the patients’ treatment course. Among all models, the top-performing one that was trained with the images across different time points of a treatment course yielded a Dice similarity coefficient of 93% and a sensitivity of 96% on baseline images. The top-performing model also produced accurate tumor size measurements, which is valuable for practical clinical applications. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, representing approximately 15% of all breast cancers and contributing to approximately 40% of breast cancer-related deaths , accuratAn important step in quantitative image analyses is tumor segmentation. The most commonly used method of tumor segmentation is manual contouring and annotation by experienced radiologists. However, this process is labor-intensive and tedious, as well as being susceptible to human errors and inter-reader variations . To overDeep learning techniques, especially convolutional neural networks , have beDynamic contrast-enhanced (DCE) MRI is capable of measuring contrast agent kinetics and has shown high sensitivity in detecting breast cancer . The kinHowever, the success rate of the translation of these models trained from one dataset to a different and independent dataset is still very limited. The technical challenge is likely caused by the methods’ configuration and parameters optimization that are needed for the dataset diversity. Adapting a model iteratively requires extensive knowledge and effort, which increases the complexity of the clinical workflow. Another challenge is the lack of high-quality datasets for the model training of a specific disease population. MRI datasets are typically much larger in image size but smaller in population size than sonogram or mammogram datasets. The availability of DCE-MRI data specific to TNBC patients is even more limited and poses another barrier to train a functional model. In this work, we aimed to develop a model for automated segmentation of TNBC on DCE-MRI images. The deep learning framework we employed to build our model is nnU-Net , which iThis study was approved by the Institutional Review Board (IRB) of The University of Texas MD Anderson Cancer Center and was part of an ongoing IRB-approved prospective clinical trial (NCT02276443) of patients with stage I-III TNBC who were being monitored for responses to neoadjuvant systemic therapy. This study followed the ethical guidelines set out in the Declaration of Helsinki, and written informed consent was obtained from each participant.A total of 301 patients with biopsy-confirmed stage I-III TNBC were included in this study. The data inclusion criteria were identical to and described in a previously published work . The imaThe DCE-MRI images were acquired using a 3T GE 750w MR scanner  and an eight-channel bilateral phased array breast coil. The imaging protocol utilized a three-dimensional (3D) T1-weighted DISCO  sequenceDCE-MRI images and binary masks were first zero-padded along both sides of the imaging volume to 192 slices, and then the full-field-of-view images were fed for model training without any cropping. Two breast radiologists with 6 years of experience (R.M.M.) and 11 years of experience (S.P.) manually segmented the tumors in consensus using an in-house MATLAB-based software . These manually segmented tumors are shown by the reference masks in Figures 2 and 3. The manual segmentation was performed on a subtraction image obtained by subtracting the pre-injection phase  from the early phase (the frame at approximately 2.5 min after injection). Voxels of high contrast uptake between phases were identified as tumors, and voxels of signal void from biopsy clips or tumor necrosis were excluded. In cases where multiple tumors were present, only the dominant one was labeled at BL, and the same tumor was followed at C2 and C4 if images were acquired at those time points. In addition, we calculated and used the following three semiquantitative maps: positive enhancement integral (PEI) , signal To evaluate the impact of central necrosis and biopsy clips on segmentation accuracy, we compared models with their inclusion and exclusion. Unless noted otherwise, the default segmentation used for the constructed models described below in We used the default original configuration of nnU-Net . The software environment included an Ubuntu Linux 18.4.6 operating system, Python 3.8.12, CUDA 11.1, cuDNN 7.6.5, and TensorFlow 2.8.0.The performance of each model was evaluated using four overlap-based segmentation metrics, true positive, false negative, false positive, and true negative, using the manually labeled mask as the reference standard on a per-subject basis. The true positive, false negative, false positive, and true negative are defined as the percentage of the number of voxels within the union between the reference and predicted masks, of the number of voxels within the reference mask but outside the predicted mask, the number of voxels within the predicted mask but outside the reference mask, and the number of background voxels outside the reference mask, respectively. The DSC and sensitivity were calculated and averaged across all subjects as the metrics for overall performance .Given the non-normal distribution of the results, the within-group results of subject-based DSC and sensitivity were summarized using interquartile and median calculations; the paired two-sample comparison was performed with Wilcoxon signed-rank test and the unpaired two-sample comparison was performed with Wilcoxon rank-sum test; the multiple comparison was performed using Kruskal–Wallis test. For all comparisons, α = 0.05 was considered the threshold for statistical significance and adjusted with Bonferroni correction for multiple comparison. The segmentation performance was examined for primary tumors with the following largest dimensions: ≤2 cm (T1), 2–5 cm (T2), and ≥5 cm (T3–4).2 value was 42.1 for DSC (p < 0.05) and 59.9 for sensitivity (p < 0.05); post hoc-paired Wilcoxon signed-rank test was performed and adjusted at p = 0.05/4 = 0.0125. For DSC, nnU-Net_BL was better than nnU-Net_PEI (p < 0.0125), nnU-Net_SER (p < 0.0125) and nnU-Net_MSI (p < 0.0125) but similar to nnU-Net_Comb (p = 0.90). For sensitivity, nnU-Net_BL was better than nnU-Net_SER (p < 0.0125) and nnU-Net_MSI (p < 0.0125) but similar to nnU-Net_PEI (p = 0.19) and nnU-Net_Comb (p = 0.58). The Kruskal–Wallis test demonstrated significant differences among models with different input metrics . The χ2 p = 0.17) in nnUnet_Excl  and nnUnet_Incl  (A Wilcoxon signed-rank test showed similar group DSC mean ranks (y clips) . The tumy clips) B. The sey clips) C.p < 0.05) and sensitivity (p < 0.05). Similarly, nnU-Net_3tpt had a better performance than nnU-Net_C2 on the testing dataset of C2 for both DSC (p < 0.05) and sensitivity (p < 0.05), and a better performance than nnU-Net_C4 on the testing dataset of C4 for both DSC (p < 0.05) and sensitivity (p < 0.05). The decrease in performance over the treatment time points in two representative subjects is shown in The nnU-Net_3tpt model, which was trained on a combination of data from all three time points, BL, C2, and C4, demonstrated better segmentation performance than the models trained on data from a single time point  . The Pai2 =15.7, p < 0.05). The DSC had higher mean ranks for tumors larger than 2 cm but smaller than 5 cm (p < 0.05) in relation to tumors smaller than 2 cm. Similarly, the tumors larger than 5 cm had a higher mean rank of DSC than tumors smaller than 2 cm (p < 0.05), as shown in p < 0.05) for tumors larger than 2 cm but smaller than 5 cm. For tumors that were 2 cm or smaller (p = 0.07) and tumors at 5 cm or larger (p = 0.29), the performance of the two models was similar. The higher mean ranks of DSC and sensitivity for nnU-Net_3tpt than for nnU-Net_C2 and nnU-Net_C4 across tumor sizes are presented in The Kruskal–Wallis test demonstrated significant differences in DSC among various tumor sizes over the test set of 3tpt (χp < 0.05) between predicted segmentation with nnU-Net_3tpt and the reference standard (p < 0.05) and 0.73 at C4 test set (p < 0.05). The tumor size of the predicted segmentation from nnU-Net_3tpt was also evaluated using the reference standard . The intstandard A, which In this study, we employed an automated deep learning framework for medical imaging segmentation, nnU-Net, in conjunction with 744 datasets to develop an accurate segmentation model specifically for TNBC under treatment. We evaluated a range of semiquantitative maps from DCE-MRI as model inputs and found that the subtraction images of the initial phases from the peak arterial phases yielded the optimal contrast for model training. Additionally, using images from multiple time points during a patient’s treatment resulted in significantly better segmentation performance than using images from a single time point during treatment. This improved performance of our top-performing model likely stemmed from a greater diversity in terms of tumor size, shape, location, signal intensity patterns, and other morphological characteristics. Notably, our model accurately estimated tumor size, which is important as it is often necessary to measure changes in tumor size during treatment. Our results indicate that DCE subtraction images provide sufficient image information to achieve good tumor segmentation with our deep learning model. To our knowledge, most reported breast cancer segmentation models for DCE-MRI use the original multiphasic images ,44 or thTo the best of our knowledge, our study is the first to perform model training using datasets including images of patients from multiple treatment time points. The nnU-Net_3tpt model trained using this approach demonstrated better performance than the model trained using data from only a single treatment time point. By combining data from three treatment time points, the training datasets were effectively tripled. Since all the model training parameters remained identical, it is possible that the increased dataset size contributed to the improved model performance. With the expanded training dataset, our nnUNet_3tpt model exhibited performance similar to or even better than that of recent breast tumor segmentation studies. For instance, when Yue et al. evaluated model performance on a dataset of 1000 subjects , their own model, Res-UNet, achieved a DSC of 0.894, and their implementation of nnU-Net achieved a DSC of 0.887 , whereasThe segmentation models using masks with or without central necrosis and biopsy clips exhibited similar DSC and sensitivity, indicating that our models based on the nnU-Net framework are flexible and stable. To the best of our knowledge, most published studies on breast tumor segmentation employed reference masks that included central necrosis and biopsy clips ,28,44,50Our nnU-Net_3tpt model had good performance but can be further improved in several aspects. First, nnU-Net_3tpt produced increasingly better segmentation results for tumors with larger sizes, which may explain the better performance at BL than at C2 and C4 because tumors at BL are untreated and tend to be larger. A similar trend was noticed in other studies ,49,51. TWe developed a fully automated, high-performance segmentation model for TNBC patients using deep learning and a large cohort of DCE-MRI images acquired longitudinally over the patients’ treatment course. Of the various types of images used for model training, we found that the simple subtraction images had the best performance. Our model was also capable of reliably segmenting tumors with either exclusion or inclusion of the central necrosis and biopsy clips. The performance of our model, especially for small tumors, may be further improved in a future investigation.


TM AIO systems in the detection of HLA-B*27 were compared. The mean absorbance  values for the MaelstromTM 8 and LabTurboTM AIO systems were found to be 1.88 and 1.9, respectively. The housekeeping gene was amplified and quantified using a real-time PCR assay across all DNA extracts to check the quality of the extracted human DNA. The results were expressed as the cycle threshold (Ct) values for all DNA extracts from both platforms. The mean Ct values for the Roche Cobas z480 and LabTurboTM AIO systems were found to be 22.7 and 20.4, respectively. This study demonstrated that the semi-automated method and the LabTurboTM AIO system yield consistent results for the detection of HLA-B*27. However, compared to the semi-automated method, the LabTurboTM AIO system provides standardized procedures, avoids manual handling, and improves turnaround time.The adoption of an automated system can decrease the hands-on time requirements in a clinical laboratory setting. For the detection of HLA-B*27, implementing a high-throughput and fully automated system has several advantages over using manual methods. Therefore, this study aimed to evaluate automation efficiency for the detection of HLA-B*27. Peripheral blood samples were obtained from 50 participants, and DNA was isolated from these samples. A Pharmigene PG27 detection kit was used for the qualitative detection of HLA-B*27. The performances of the semi-automated and fully automated LabTurbo The advancement in molecular diagnostics has facilitated the genetic testing of genetic diseases as well as the identification of multiple pathogens. The operation of these tests requires extensive training in molecular biology. The implementation of laboratory automation can simplify the process of analyzing multiple specimens, thereby increasing the throughput of specimen testing, standardizing the performance of molecular testing procedures, shortening testing turnaround times, and reducing labor time and cost. In essence, automation combines laboratory robotics and analytical software. Nowadays, automation is becoming more sophisticated than simply facilitating the integration of sample extraction, as it also supports nucleic acid amplification and detection. A fully automated molecular diagnostic system is a robust and useful system that is capable of a broad variety of applications ,2.As molecular diagnostics is a complex process involving several procedures from nucleic acid extraction to result interpretation, technologists can only process a few clinical samples at a time. Moreover, when high-volume testing is demanded, higher manpower costs are incurred in the long run. Additionally, regular laboratory testing is highly labor-intensive and may lead to manual errors, which can result in poor patient outcomes. If the number of clinical samples to be processed by laboratories increases dramatically, productivity has to be improved. Therefore, automated equipment such as robotic liquid-handling platforms can replace manual processes in molecular diagnostics workflows. This will reduce hands-on time and improve accuracy in a clinical laboratory setting. Bioinformatic software also facilitates standardized reporting and the interpretation of laboratory test results.TM AIO  COVID-19 RNA testing kit is a feasible and reliable method for SARS-CoV-2 detection [TM AIO extraction system, which is combined with a real-time reverse transcription polymerase chain reaction (rRT-PCR) platform. According to them, the pooling strategy applied in the LabTurboTM AIO system resulted in zero volume loss for SARS-CoV-2 detection using rRT-PCR [TM AIO system offered a high-throughput detection of SARS-CoV-2 variants, influenza A/B, and respiratory syncytial virus, along with shortening the duration of the entire process from its initiation to completion [TM AIO system had not been used for the detection of HLA-B*27. Recently, Chang et al. reported that the LabTurboetection . They de rRT-PCR . Chung empletion . Until rThe human leukocyte antigen (HLA) system is a gene complex encoding the major histocompatibility complex proteins in humans. These cell-surface proteins are in charge of the regulation of the immune system. Classic class I HLA consists of HLA-A, -B, and -C genes, whereas the main class II HLA genes are DP, DQ, and DR . StudiesIn the present study, we aimed to evaluate the efficiency of automation in the detection of HLA-B*27.2EDTA. One part of each sample was analyzed using a semi-automated procedure, whereas the other part was analyzed using a fully automated procedure. Ethical approval was obtained from the Institution Review Board of China Medical University Hospital (CMUH112-REC2-026) and (CMUH112-REC2-026 (AR-1)). The requirement for informed consent from each patient was waived.The present study included a total of 50 participants who had requested HLA-B*27 typing. Out of the 50 samples that were tested, 35 (70%) were obtained from male patients while 15 (30%) were obtained from female patients. The median age of these 50 patients was 47.5 years (12–69). Peripheral blood specimens (10 mL) were collected in KTM 8 Autostage  was used for extracting DNA from the whole blood samples using the TANBead® nucleic acid extraction kit , as per the manufacturer’s instructions. DNA was extracted from samples of 300 μL volume. The MaelstromTM 8 Autostage is a high-efficiency purification system driven by TANBead’s patented technology [TM 8 Autostage, a maximum of eight samples can be processed during each run.In this study, the Maelstromchnology . With TATM AIO system  was used for extracting DNA from the whole blood samples using the LabTurbo Viral DNA/RNA Mini Kit , as per the manufacturer’s instructions. The total DNA extracted from the whole blood sample (50 µL) was a final eluate volume of 60 µL. With the LabTurboTM AIO system, a maximum of 48 samples can be processed during each run. The purity of the extracted DNA was determined using a spectrophotometer. We used the HLA-B*27 detection kit  to the LabTurboTM AIO system, using specific primers and probes to detect the HLA-B*27 allele. The HLA-B*27 detection kit  was handled according to the manufacturer’s instructions. The sensitivity and specificity of the HLA-B*27 detection kit were greater than 99%, which indicates the high sensitivity of major HLA-B*27 subtypes, including HLA-B*2701~2707. The procedures followed for the LabTurboTM AIO system are presented in the flow chart in the The LabTurboTM AIO system, each 25 μL reaction mixture contained 12.5 μL of 2 × PCR master mix, 2.5 μL of primer/probe mixture, 5 μL of RNase-free water, and 5 μL of extracted RNA. HLA-B*27 was detected using the following thermal cycling conditions: 95 °C for 10 min, 35 cycles at 95 °C for 15 s, and 71 °C for 60 s .For analysis on the LabTurboFor analysis on the Roche Cobas z480 system , each 25 μL reaction mixture contained 12.5 μL of 2 × PCR master mix, 2.5 μL of primer/probe mixture, 8 μL of RNase-free water, and 2 μL of extracted RNA. HLA-B*27 was detected using the following thermal cycling conditions: 95 °C for 10 min, 35 cycles at 95 °C for 15 s, and 71 °C for 60 s .The software used for the statistical analyses was SPSS version 22.0  for Windows. The detailed results are represented in TM 8 and LabTurboTM AIO systems were observed to be 1.88 and 1.9, respectively. The housekeeping gene was amplified and quantified using a real-time PCR assay in all of the DNA extracts to check the quality of the extracted human DNA, and the results were expressed as the cycle threshold (Ct) values for all DNA extracts from both platforms. The mean Ct values for the Roche Cobas z480 system  and the LabTurboTM AIO system were 22.7 and 20.4, respectively. TM 8 and LabTurboTM AIO systems obtained by analyzing the clinical samples.The mean absorbance  values for the MaelstromTM AIO system obtained a positive percent agreement of 100% as well as a negative percent agreement of 100% by using a semi-automated assay  for genotyping HLA-B*27. The authors concluded that PCR-RFLP demonstrated robustness, technical simplicity, and cost-effectiveness . HoweverTM 8 Autostage (Taiwan Advanced Nanotech) utilizes a magnetic-bead-based system to extract nucleic acids. This system is designed to be easily used as it can process up to eight samples simultaneously. Our previous study reported that the MaelstromTM 8 Autostage offers standardized procedures, the avoidance of sample-to-sample cross contaminations, ease of use, improvement in turnaround time, and lesser hands-on time compared to the manual extraction method  [DNA extraction has evolved from solution- and solid-phase manual techniques, which were initially performed manually, to incorporate automated methods. The use of high-quantity and high-quality DNA is an important aspect of downstream applications. Therefore, nucleic acid extraction is an important step in laboratory procedures, which are required to carry out further molecular research applications. It is necessary to select a feasible and reliable extraction method, and there are a few things to consider when estimating the available options. These considerations may include cost-effectiveness, technical requirements, time efficiency, and storage requirements . The Maeini Kit) .TM AIO system has been designed as a fully automated and enclosed instrument to eliminate manual handling. In the present study, we demonstrated that the mean absorbance  values for the MaelstromTM 8 and LabTurboTM AIO systems were 1.88 and 1.9, respectively. Further, the housekeeping gene was amplified during the real-time PCR reaction as an internal control for the extraction method. The mean Ct values for the Roche Cobas z480 and LabTurboTM AIO systems were 22.7 and 20.4, respectively  on a 96-well plate, whereas we only required 25 min to load DNA samples and reagents (Pharmigene PG27) on a 96-well plate on the LabTurboTM AIO system.After comparing the results of the semi-automated and fully automated assays, we found that the fully automated assay was superior in detecting HLA-B*27. Further, we found that the use of the LabTurboTM AIO system.A major concern in the implementation of a fully automated assay is the potential for the cross-contamination of negative specimens as a consequence of aerosolization, faulty robotics, or robotic errors during the process of nucleic acid extraction for applications in amplification analysis . HoweverTM AIO system offered a high-throughput detection of pathogens with a short turnaround time [TM AIO system utilizes a patented “membrane tube vacuum flow extraction technology” to improve detection sensitivity by producing high-purity and high-yield total nucleic acid [TM AIO system can handle 864 samples per day in a continuous operation mode [TM AIO system. The main strength of our study is that it evaluates the performance of the LabTurboTM AIO system for the detection of HLA-B*27, whereas its main drawback is that it did not obtain clinical information about the patients from the physical examination center and, therefore, could not determine the frequency of HLA-B*27 in central Taiwan. Future studies should aim to overcome this limitation by including patients from the outpatient department. As mentioned above, the multiplex RT-PCR implemented on the LabTurbound time . The Labeic acid . Recentlion mode . To the TM AIO system is a fast and reliable technique for HLA-B*27 detection during routine laboratory testing. We have demonstrated the feasibility and reliability of a fully automated molecular analysis of HLA-B*27 with high throughput and significantly less hands-on time by using the LabTurboTM AIO system.In summary, the LabTurbo


Lymphoepithelioma-like intrahepatic cholangiocarcinoma (LEL-ICC) is a rare variant of intrahepatic cholangiocarcinoma. Epstein–Barr virus (EBV) infection was considered to play a pivotal role in the tumorigenesis of LEL-ICC. It is difficult to diagnosis of LEL-ICC due to the lack of specific features regarding the laboratory test results and imaging findings. At present, the diagnosis of LEL-ICC mainly depends on the histopathologic and immunohistochemical examinations. In addition, the prognosis of LEL-ICC was better than classical cholangiocarcinomas. To our knowledge, only few cases of LEL-ICC have been reported in the literature.We presented a case of a 32-year-old Chinese female with LEL-ICC. She had a 6-month history of upper abdominal pain. The magnetic resonance imaging (MRI) showed a 1.1× 1.3 cm lesion in the left lobe of liver, appearing low signal intensity on T1-weighted images and high signal intensity on T2-weighted images. The patient underwent laparoscopic left lateral sectionectomy. The postoperative histopathologic and immunohistochemical examinations results allowed for the definitive diagnosis of LEL-ICC. The patient was free from tumor recurrence after a 28 months follow-up.In this study, we reported a rare case of LEL-ICC associated with both HBV and EBV infection. EBV infection might play a pivotal role in the carcinogenesis of LEL-ICC, and surgical resection is still the most effective treatment at present. Further research on the etiology and treatment strategies of LEL-ICC is required. Epstein–Barr virus (EBV) belongs to the herpesvirus family with oncogenic properties . EBV inf9/L (3.5-9.5), platelets 249×109/L (100–300), total bilirubin 12.5 umol/L (5.0-28), and AST 35 IU/L (<50). Serological testing for tumor marker of carcinoembryonic antigen (CEA) was 5.54 ng/ml  and hepatitis B surface antigen (HBsAg) was positive. The hepatitis B virus DNA (HBV-DNA) was less than 1×102 IU/ml (HBV-DNA ≥ 1×102 IU/ml was defined as HBV infection active), suggesting that HBV infection was inactive. The cancer antigen19-9 , CA125  and α-fetoprotein  was 25.6 U/ml, 13.3U/ml and 3.37, respectively. Abdominal computed tomography (CT) showed the lesion in the left lobe of liver was detected, and no tumor was detected in any other organs , white blood cells 7.12×10Post-operative recovery of the patient was well. The patient was discharged on postoperative day 5 with good general condition. The laboratory parameters were normal and we recommended regular follow-up in the outpatient clinic.via blood examination, ultrasonography (US), CT, and MRI. The systematic update of patients’survival information was performed once a year. The last outpatient follow-up was in August 2022, and the tumor markers were normal. The patient was free from tumor recurrence after a 28 months follow-up   . Previouization) , 3.A recent review by Ding et al.  identifiClinical presentations of LEL-ICC are nonspecific. Most patients were almost asymptomatic, and the hepatic lesion was identified during a routine health examination. Some patients have the upper abdominal pain, abdominal fullness or fever . The defThe prognosis of LEL-ICC was better than classical cholangiocarcinomas . NotableIn this study, we reported a rare case of LEL-ICC associated with both HBV and EBV infection. The patient underwent laparoscopic left lateral sectionectomy based on the preoperative diagnosis of HCC, and LEL-ICC was confirmed by histopathologic and immunohistochemical examinations. EBV infection might play a pivotal role in the carcinogenesis of LEL-ICC, and surgical resection is still the most effective treatment at present. In addition, the favorable prognosis could be generally achieved after radical resection. Further research on the etiology and treatment strategies of LEL-ICC is required.The original contributions presented in the study are included in the article/Written informed consent was obtained from the individual (s) for the publication of any potentially identifiable images or data included in this article.FL and QX contributed to the data acquisition and drafted the manuscript. PR contributed to data acquisition. Y-XL and F-YL contributed to the study design and revised the manuscript. All authors contributed to the article and approved the submitted version.


The mononuclear cells were collected from rat BM using density gradient centrifugation and MSCs and C-kit+ HSCs were isolated. Then, cells were divided into two groups and differentiated into granulocytes; C-kit+ HSCs alone (control group) and co-cultured C-kit+ HSCs with MSCs . Subsequently, the granulocyte-differentiated cells were collected and subjected to real-time PCR and Western blotting for the assessment of their telomere length (TL) and protein expressions, respectively. Afterwards, culture medium was collected to measure cytokine levels. CD34, CD16, CD11b, and CD18 granulocyte markers expression levels were significantly increased in the experimental group compared to the control group. A significant change was also observed in the protein expression of Wnt and β-catenin. In addition, MSCs caused an increase in the TL of granulocyte-differentiated cells. MSCs could affect the granulocyte differentiation of C-kit+ HSCs via increasing TL and Wnt/β-catenin protein expression.Mesenchymal stem cells (MSCs) are effective in hematopoietic engraftment and tissue repair in stem cell transplantation. In addition, these cells control the process of hematopoiesis by secreting growth factors and cytokines. The aim of the present study is to investigate the effect of rat bone marrow (BM)-derived MSCs on the granulocyte differentiation of rat BM-resident C-kit Br+ HSCs alone), and experimental group (co-cultured C-kit+ HSCs with MSCs).The cells were divided into the two groups: control group  were subjected to the SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Next, membranes were incubated with primary antibodies β-actin , CD34 , CD16 , CD11b , CD18 , Wnt , and β-catenin , and incubated with secondary antibodies (1:5000). Antigen-antibody complex were detected by enhanced chemiluminescence (ECL) reagent. β actin was used as the loading control ,28.2.7+ HSCs were collected and the aTL was measured by real-time PCR as previously described by Fathi and Farahzadi (2022)  through to 10−6 [1.18 × 103] dilution) were performed.A telomere standard curve was created by the dilution of known quantities of a synthetic 84-mer oligonucleotide containing TTAGGG only. For generating a standard curve, the serial dilutions of TEL STD A (102.7.2−1 through to 10−6 dilution) were performed.SCG was used for determining the genome copies per sample. For generating a standard curve, the serial dilutions of SCG STD A  was performed . In brief, a 96-well plate was coated with detection Reagent A for 16 h at 4 °C. Then, cell culture media were added into the 96-well plate, which had been coated with IL-3 , IL-4 , and IL-6  antibodies, and detected via the ELISA sandwich technique.2.9The results were analyzed using the software program Graph Pad Prism version 6.01. T-test and two-way ANOVA were used to determine the significant difference among groups.33.1As shown in 3.2+ cells enrichment was assessed by ICC and flow cytometry. As shown in + cells had high (96.9%) C-kit expression levels. + cells (blue dots) from the isotype control population (red blots).The purity of the rat BM-resident C-kit3.3+ HSCs were collected and granulocyte cell markers were analyzed by western blot as shown in P < 0.01). Also, the Wnt and β-catenin signaling pathways were investigated. As shown in P < 0.01 and ∗P < 0.05, respectively).Following the co-culture, granulocyte differentiated C-kit3.4+ cells. Furthermore, the expression of TERT protein in the experimental group was significantly (0.47-fold) decreased when compared to the control group  increased in experimental group compared to the control group  that may affect the granulocyte differentiation. These measurements revealed that the secretion of IL-3, IL-4, and IL-6 were significantly  or CSFs that regulate hematopoiesis . It was + HSCs were isolated from rat BM. After culturing and characterizing these cells, the interaction between MSCs and C-kit + HSCs was investigated under in vitro co-culture conditions. At the end of the co-culture period, the granulocyte differentiated rat BM-derived C-kit+ cells were collected and subjected to the Western blot analysis and real-time PCR for investigating the granulocyte marker expression, Wnt/β-catenin signaling components and aTL measurement, respectively. Accordingly, CD11b surface marker is expressed on the cell surface of monocytes and neutrophils. This surface marker provides the possibility of other factors to bind the granulocyte growth factors by specific ligands such as C3bi, fibrinogen, intracellular adhesion molecules-1 (ICAM-1), and Factor x. Additionally, it prepares neutrophil cells for phagocytosis and so leads monocyte/macrophage and T-cells to express immune responses to damages. The detection of CD11b in tissues and blood is an evidence supporting the proper function of immune T-cells and monocyte/macrophage cells in countering inflammation and viral attacks [+ cells from the BM.In the present study, the MSCs and C-kit attacks . It has 35. CD16 is an integrin expressed on the surface of neutrophils, monocyte/macrophage, and a particular type of lymphocytes, i.e., NK cells. This surface marker determines the cell function and cellular distribution. CD16 presence on the neutrophil surface is a known maturity indicator and its absence is a sign of leukocytosis and tissue necrosis [Integrins mediate crucial adhesive recognitions through its ability in binding to multiple unrelated ligandsnecrosis .trans-membrane receptors, named CDlla/CD18 (LFA-l), CDllb/CD18 (Macl and CDllb), and CD11c/CD18. These are broadly distributed on all leukocytes of lymphoid and myelomonocytic lineage.Yet another leukocyte integrin, CD18, consists of three heterodiameric + cells include CFU-M, CFU-G, CFU-GM, BFU-E, which are particularly rich in the primary types of colony-forming cells such as CFU-mix and CFU-blast36. This maker as BM harvested hematopoietic cells is clinically used for transplantation and gene therapy studies. These cells have accelerated studies focused on the developmental hematopoiesis.CD34 is a surface glycol phosphoprotein expressed on progenitor cells, small-vessel endothelium, and embryonic fibroblasts. CD34+/CD38−Lin- populations [Flores-Guzman et al. in their study showed that rat BM-derived MSCs play a key role in primitive cell phenotype maintenance in CD34ulations .Emerging evidence generally supports the belief that cell differentiation often coincides with the activation or suppression of the signaling pathway. The importance of Wnt signaling in hematopoietic development has been reported previously. Activation of the Wnt pathway increased the number of HSCs as identified by colony forming cell assay ,20.+ cells and the results demonstrated that MSCs cause a significant increase in the expression levels of CD34, CD16, CD11b, and CD18 surface markers of rat BM-derived C-kit+ cells by about 2.77, 2.67, 3.08, and 3.18 folds, respectively. Likewise, in the previous study conducted by Fathi et al. (2021), it was indicated that MSCs cause to increase in the CD34, CD16, CD11b, and CD18 surface markers. In addition, in the study by Nikkhah et al. (2018), it was pointed that MSCs cause an increase in the CD11b surface marker as well as some granulocyte subset-specific genes such as PU.1, CD11b, lysozyme, MPO, CD64 that lead to an increase in the HL-60 leukemic cells [In a study by Fathi et al. (2021), it was reported that co-culturing of MNCs with rat BM-derived MSCs cause an increase in the expression levels of granulocyte markers of MNCs, e.g., CD34, CD16, CD11b, and CD18 . Based oic cells . As well+ HSCs cells.Accordingly, our results are in line with those of the previous studies regarding the important role of cytokines in the granulocyte differentiation of C-kit+ HSCs will have to be undertaken to understand better these processes. TL shortening is known as one of the key characteristics of cell differentiation in cell therapy. Some studies have shown that aging is associated with the loss of TL in HSCs. In this study, our findings on aTL measurement of granulocyte-differentiated C-kit+ HSCs supported the previous findings of other investigations, showing that the differentiated cells have the shortest TL.Further studies on molecular regulation of the granulocyte-differentiated C-kit+ HSCs. This finding brings a new insight into the importance of Wnt/β-catenin signaling pathway during this differentiation process.In conclusion, our study for the first time demonstrated that rat BM-derived MSCs play an important role in promoting the granulocyte differentiation of rat BM-derived C-kitE.F. as the executive of the project had main contribution in conception and design, data analysis, and manuscript writing; I. V. as the main colleague of the project in providing the kits and some materials needed for this project as well as interpretation of experimental procedure; R.F. and S.A.M − N as colleagues involved in performance of experiments, data analysis and manuscript writing.Center for International Scientific Studies & Collaborations (CISSC), Ministry of Science Research and Technology of Iran.This work has been supported by the The ethical code was obtained from the Committee on the Ethics of Tabriz University of Medical Sciences (Ethics No. IR. TBZMED.VCR.REC.1397.322).All of the experimental procedures involving animals were conducted in accordance with the ethics committee of Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC.1397.322) approved protocols. This article does not contain any studies with human subjects.There are no human subjects in this article and informed consent is not applicable.The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.The authors declare that they have no conflict of interest.


Staphylococcus aureus are ubiquitous Gram-positive pathogenic bacteria and are rapidly acquiring antibiotic resistance. Here, celecoxib was encapsulated into cubosomal nanoparticles, and the particle morphology, size distribution, zeta potential, entrapment efficiency, and celecoxib release were evaluated in vitro. Also, a systemic infection model in mice elucidated the in vivo antibacterial action of the celecoxib cubosomes. Cubosomes are a nanotechnology-based delivery system which can adhere to the external peptidoglycan layers of Gram-positive bacteria and penetrate them. The size distribution investigation revealed that the prepared celecoxib-loaded cubosomes had a mean particle size of 128.15 ± 3.04 nm with a low polydispersity index of 0.235 ± 0.023. The zeta potential measurement showed that the prepared cubosomes had a negative surface charge of −17.50 ± 0.45, indicating a highly stable nanodispersion formation with little susceptibility to particle aggregation. The cubosomal dispersion exhibited an entrapment efficiency of 88.57 ± 2.36%. The transmission electron micrograph for the prepared celecoxib-loaded cubosomes showed a narrow size distribution for the cubosomal nanoparticles, which had a spherical shape and were non-aggregated. The tested cubosomes diminished the inflammation in the treated mice’s liver and spleen tissues, as revealed by hematoxylin and eosin stain and Masson’s trichrome stain. The immunostained tissues with nuclear factor kappa B and caspase-3 monoclonal antibodies revealed a marked decrease in these markers in the celecoxib-treated group, as it resulted in negative or weak immunostaining in liver and spleen that ranged from 4.54% to 17.43%. This indicates their inhibitory effect on the inflammatory pathway and apoptosis, respectively. Furthermore, they reduced the bacterial burden in the studied tissues. This is alongside a decrease in the inflammatory markers  determined by ELISA and qRT-PCR. The IL-1β levels were 16.66 ± 0.5 pg/mg and 17 ± 0.9 pg/mg in liver and spleen, respectively. Also, IL-6 levels were 85 ± 3.2 pg/mg and 84 ± 2.4 pg/mg in liver and spleen, respectively. In conclusion, the current study introduced cubosomes as an approach for the formulation of celecoxib to enhance its in vivo antibacterial action by improving its oral bioavailability.There is a great need for novel approaches to treating bacterial infections, due to the vast dissemination of resistance among pathogenic bacteria.  Staphylococcus aureus is a Gram-positive bacterium that belongs to the normal flora. It is an opportunistic pathogen that can result in various illnesses ranging from mild skin infections to life-threatening systemic ailments [ailments . These bailments ,3. In adailments . Thus, tailments ,5,6.Drug repurposing or drug repositioning means the identification of novel therapeutic activities from the currently existing FDA-approved drug molecules that are used clinically. It is a promising approach for treating various human ailments like bacterial infections, as the currently approved drugs used to treat different diseases are investigated for their possible antibacterial activity ,8. This Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed classes of drugs. They possess long-standing and wide-ranging therapeutic applications, from the salicylate-containing willow leaves to the recent highly selective cyclooxygenase-2 (COX-2) inhibitors. Celecoxib is one of the non-steroidal anti-inflammatory compounds that are widely employed to treat symptoms of inflammation like arthritis .Celecoxib is a poorly water-soluble, very lipophilic drug that has a poor oral bioavailability ranging between 22% and 40% when administered in the form of traditional capsules . It showWith the growing concern about antimicrobial resistance and the need for novel therapeutic strategies, repurposing existing drugs has emerged as a promising approach. Celecoxib has recently gained attention for its potential antibacterial activity ,20. HoweThe development of nanoformulations has opened new avenues for enhancing the antibacterial activity of different antibiotics, including cephradine ,22, cefaCubosomes can cling to the external peptidoglycan layers of Gram-positive bacteria and then penetrate the bacterial cell. The contact in Gram-negative bacteria occurs in a two-stage process involving fusion with the outer lipid membrane and subsequent diffusion through the inner wall. The inherent self-assembly property of cubosomes confers upon them a unique potential to facilitate the transportation of payloads via the process of membrane fusion . The fusS. aureus through a systemic infection model in mice, using different histological, immunohistochemical, and molecular techniques. The cubosomal nanodispersion is expected to enhance the antibacterial efficacy of celecoxib and overcome the challenges associated with bacterial resistance, thereby providing a potential alternative to current antibiotics, which suffer from resistance.Here, we aimed to develop celecoxib in cubosomal NPs and evaluate the influence of cubosomal formulation on the in vivo antibacterial efficiency of celecoxib against The prepared celecoxib-loaded cubosomal NPs were assessed for particle size, surface charge, morphology, drug loading%, and EE%. The size distribution investigation revealed that the prepared celecoxib-loaded cubosomes had a mean particle size of 128.15 ± 3.04 nm with a low polydispersity index of 0.235 ± 0.023 A, which The transmission electron micrograph A of the p ˂ 0.05) higher % cumulative celecoxib release  compared to 61.89 ± 4.16% for the plain celecoxib suspension. The higher drug release from the cubosomal dispersion may be due to the developed small particles in a nanometric range with a large surface area [2 of 0.997 ± 0.054, demonstrating that diffusion is the dominant mechanism of release. These findings agree with previous observations on the release of drugs from cubosomes [The release profiles of the plain celecoxib suspension and celecoxib-loaded cubosomes are shown in ace area . The kinubosomes ,39,40.S. aureus in vitro. Interestingly, the celecoxib (free drug) and the celecoxib cubosomes revealed antibacterial activity, shown by the inhibition zones around the wells  stain, as shown in H&E staining of liver and spleen sections of group I showed normal features of the central vein with an average-sized portal tract confined by hepatocytes cords and isolated by blood sinusoids. The spleen had a white pulp of normal size (lymphoid follicles), and the central arteriole was bounded by red pulp that had average size. Conversely, group III revealed focal inflammation in both liver and spleen tissues. The histological features were improved in group IV as there was a normal portal tract bounded by normal cords of hepatocytes and few inflammatory cells. Spleen tissues revealed normal-sized red pulps bounded by normal-sized white pulp.Masson’s trichrome stain was used to investigate the fibrosis of liver and spleen sections. The group treated with celecoxib cubosomes revealed a noticeable decline of the collagen fibers compared to the normal group.The immunohistochemical studies elucidated the immunostaining of the liver and spleen tissues with caspase-3 and nuclear factor kappa β (NF-kβ), as shown in Apoptosis and inflammation usually accompany systemic infections. Thus, the drugs which inhibit both processes by inhibiting the enzymes and mediators involved in them are very beneficial in inhibiting the pathogenesis of bacterial infections . Thus, iCaspase-3 is an enzyme that is involved in apoptosis or programmed cell death , and it Regarding NF-kβ, it mediates the inflammatory process by induction of the release of the proinflammatory mediators ,46. HereThe levels of the interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) were determined in the liver and spleen of the experimental groups using ELISA, as displayed in Cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-α) gene expression were analyzed in the liver and spleen of the experimental groups using qRT-PCR, as displayed in p < 0.05) in the inflammatory markers, including IL-1β, IL-6, COX-2, and TNF-α compared with celecoxib-free drugs. This could be explained by the cubosomal NPs enhancing celecoxib bioavailability. COX-2 inhibition was reported to improve the clearance of Burkholderia pseudomallei and Pseudomonas aeruginosa from in vivo models of infected mice [Celecoxib is an anti-inflammatory drug that works by inhibiting COX-2 enzyme activity. It could be beneficial in treating bacterial infections, as inflammation usually accompanies viral and bacterial infections ,48. Hereted mice . Also, ited mice , IL-6 [5ted mice , and TNFted mice , increasCelecoxib was obtained from Merck . Monoolein (GMO) was received as a gift from Kerry Ingredients & Flavours . Poloxamer 407 (P 407), cellulose dialysis membrane , and methanol (HPLC-grade) were obtained from Merck, .Celecoxib-loaded cubosomal dispersions were formed, as previously explained . BrieflySize dispersion and zeta potential of celecoxib-loaded cubosomes were explored using a Zetasizer Nano ZS  after appropriate dilution and analyzed in triplicate at 25 ± 0.5 °C .Cubosomes’ morphology was assessed using a transmission electron microscope (TEM) . A droplet of the suspension was adsorbed on a carbon-covered copper grid and then air-dried. Before analysis, samples were stained with a 1% sodium phosphor tungstate solution.0). The free unentrapped celecoxib amount (Q1) was calculated by centrifugal filtration of the diluted sample at 6000 rpm for 15 min. The amount of celecoxib in the attained supernatant was measured using a UV-visible spectrophotometer  at a maximum wavelength of 254 nm [2 of 0.998. The EE% was calculated according to the following equation:The entrapment efficiency (EE%) of the prepared celecoxib-loaded NPs was evaluated after separating the unentrapped free drug using the centrifugation ultrafiltration approach. Briefly, the prepared dispersion (1 mL) was diluted correctly, and then the diluted sample was mixed with methanol to dissolve cubosomes and achieve the total amount of celecoxib  at different times . The growth curve was constructed by plotting log OD620 against time (h).The impact of celecoxib and its cubosomal NPs on the growth of the bacterial isolate was revealed at 0.5 MIC values, as previously reported . The optForty mice were acquired from the faculty of veterinary medicine at Cairo University, Cairo, Egypt. All standard conditions for feeding and preserving animals were followed as previously described . The proS. aureus was explored in vivo. The bacterial suspension (107 colony forming unit (CFU)/mL) was injected for two days [The antibacterial efficiency of the celecoxib cubosomal formulation against two days  to inductwo days , group Itwo days . According to the previously reported method , the hisThe levels of the inflammatory markers (IL-1β and IL-6) in pg/mg protein were determined using an ELISA kit .The relative gene expression of the COX-2 and TNF-α were determined in the liver and spleen tissues of the different tested groups. Total RNA was extracted using the Purelink™ RNA kit , and then converted into cDNA by the Power™ cDNA kit  to synthesize the cDNA. Rotor-Gene Q 5plex  was utilized in the qRT-PCR run, and the employed primers are revealed in p < 0.05. The software utilized was Prism version 8 .Data were presented as mean ± standard deviation, and ANOVA was employed to study the experimental group differences. The difference was regarded to be significant if S. aureus. In vitro, celecoxib-loaded cubosomes entrapped 88.57 ± 2.36% of the drug and demonstrated a spherical shape using TEM with a narrow nano-size distribution. Celecoxib cubosomes led to an enhancement of the in vivo antibacterial potential against S. aureus isolate in comparison with the celecoxib-free drug. This was revealed by improved histological features using H&E as well as Masson’s trichrome stains. Also, there was an improvement in the immunohistochemical features. The celecoxib-loaded formulation exhibited a significant decrease in the inflammatory biomarkers (IL-1β and IL-6) using ELISA. In addition, qRT-PCR was employed, and it revealed a downregulation of the genes encoding COX-2 and TNF-α in the celecoxib-formulation-treated group. Thus, to the best of our knowledge, this is the first study that presented cubosome nanovesicles as an optimized formula for the delivery of repurposed celecoxib for the treatment of systemic S. aureus infection. This study presents a solution to the increasing resistance of S. aureus bacteria. In future studies, the antibacterial action of celecoxib should be investigated on other resistant bacterial pathogens, alone and in combination with different antibiotics.This work aimed to develop celecoxib-loaded cubosomes and evaluate the in vivo antibacterial action of the cubosomal formulation against